Aims. In Asia and the Middle-East, people often flex their knees deeply in order to perform activities of daily living. The purpose of this study was to investigate the 3D kinematics of normal knees during high-flexion activities. Our hypothesis was that the femorotibial rotation, varus-valgus angle, translations, and kinematic pathway of normal knees during high-flexion activities, varied according to activity. Materials and Methods. We investigated the in vivo kinematics of eight normal knees in four male volunteers (mean age 41.8 years; 37 to 53) using 2D and 3D registration technique, and modelled the knees with a computer aided design program. Each subject squatted, kneeled, and sat cross-legged. We evaluated the femoral rotation and varus-valgus angle relative to the tibia and anteroposterior translation of the medial and lateral side, using the transepicodylar axis as our femoral reference relative to the perpendicular projection on to the tibial plateau. This method evaluates the femur medially from what has elsewhere been described as the extension facet centre, and differs from the method classically applied. . Results. During squatting and kneeling, the knees displayed femoral external rotation. When sitting cross-legged, femurs displayed internal rotation from 10° to 100°. From 100°, femoral external rotation was observed. No significant difference in varus-valgus angle was seen between squatting and kneeling, whereas a varus position was observed from 140° when sitting cross-legged. The measure kinematic pathway using our methodology found during squatting a medial pivoting pattern from 0° to 40° and bicondylar rollback from 40° to 150°. During kneeling, a medial pivot pattern was evident. When sitting cross-legged, a lateral pivot pattern was seen from 0° to 100°, and a medial pivot pattern beyond 100°. Conclusion. The kinematics of normal knees during high flexion are variable according to activity. Nevertheless, our study was limited to a small number of male patients using a different technique to report the kinematics than previous publications. Accordingly, caution should be observed in generalizing our findings. Cite this article: Bone Joint J 2018;100-B:50–5

Osteoarthritis (OA) is a degenerative disease that lacks regenerative treatment options. Current research focuses on mesenchymal stem cells (MSCs) and Platelet-Rich Plasma (PRP) as regenerative therapies, but extracellular vesicles (EVs) have shown to be more advantageous. This study compares the regenerative potential of human umbilical cord MSC-derived EVs (cEVs) and platelet-derived EVs (pEVs) in ex vivo and in vivo OA models. In the ex vivo study, OA conditions were induced in human cartilage explants, which were then treated either with pEVs or cEVs. Results showed a higher content of DNA and collagen in the pEVs group compared to control and cEVs groups, suggesting that pEVs could be a potential alternative to cEVs. In the in vivo study, an OA model was established in the knee joints of rats through MIA (monoiodoacetate) injection and then treated either with pEVs or cEVs. Results showed that pEVs-treated knee joints had better subchondral bone integrity and greater OA reversion, particularly in female rats, indicating that pEVs are a viable regeneration treatment for OA and outperform cEVs in terms of efficacy. Overall, the study demonstrates the potential of EVs as a regenerative treatment for OA, with pEVs showing promising results in both ex vivo and in vivo models. The use of pEVs in clinical practice could provide a faster path to translation due to the established use of platelet concentrates in therapeutics. However, further studies are needed to fully evaluate the potential of pEVs for OA treatment and to elucidate the mechanisms behind their regenerative effects. Acknowledgments: The authors thank Dr Fernando Hierro (UIB) for their technical contribution with TEM, Mª Trinidad García (UIB) for the access to radioactivity facilities, Aina Arbós (IUNICS) for her contribution in the histology staining, María Tortosa (IdISBa) for her assistance with the animal care and ADEMA School of Dentistry for the access to the cone beam computed tomography (CBCT). Funding: This research was funded by Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, co-funded by the ESF European Social Fund and the ERDF European Regional Development Fund (MS16/00124; CP16/00124), PROGRAMA JUNIOR del proyecto TALENT PLUS, construyendo SALUD, generando VALOR (JUNIOR01/18), financed by the sustainable tourism tax of the Balearic Islands; the Direcció General d'Investigació and Conselleria d'Investigació, Govern Balear (FPI/2046/2017); the Mecanisme de Recuperació i Resiliència, intended to execute research projects of «Noves polítiques públiques per a un mercat de treball dinàmic, resilient i inclusiu», collected in Pla de Recuperació, Transformació i Resiliència, financed by European Union-Next Generation EU and driven by SOIB and Conselleria de Fons Europeus, Universitat i Cultura i la Conselleria de Model Econòmic, Turisme i Treball (NG0421) and the grant SYN20/03 from IdISBa

Aims. To fully verify the reliability and reproducibility of an experimental method in generating standardized micromotion for the rat femur fracture model. Methods. A modularized experimental device has been developed that allows rat models to be used instead of large animal models, with the aim of reducing systematic errors and time and money constraints on grouping. The bench test was used to determine the difference between the measured and set values of the micromotion produced by this device under different simulated loading weights. The displacement of the fixator under different loading conditions was measured by compression tests, which was used to simulate the unexpected micromotion caused by the rat’s ambulation. In vivo preliminary experiments with a small sample size were used to test the feasibility and effectiveness of the whole experimental scheme and surgical scheme. Results. The bench test showed that a weight loading < 500 g did not affect the operation of experimental device. The compression test demonstrated that the stiffness of the device was sufficient to keep the uncontrollable motion between fracture ends, resulting from the rat’s daily activities, within 1% strain. In vivo results on 15 rats prove that the device works reliably, without overburdening the experimental animals, and provides standardized micromotion reproductively at the fracture site according to the set parameters. Conclusion. Our device was able to investigate the effect of micromotion parameters on fracture healing by generating standardized micromotion to small animal models. Cite this article: Bone Joint Res 2021;10(11):714–722

Aims. There is a considerable challenge in treating bone infections and orthopaedic device-associated infection (ODAI), partly due to impaired penetration of systemically administrated antibiotics at the site of infection. This may be circumvented by local drug administration. Knowledge of the release kinetics from any carrier material is essential for proper application. Ceftriaxone shows a particular constant release from calcium sulphate (CaSO. 4. ) in vitro, and is particularly effective against streptococci and a large portion of Gram-negative bacteria. We present the clinical release kinetics of ceftriaxone-loaded CaSO. 4. applied locally to treat ODAI. Methods. A total of 30 operations with ceftriaxone-loaded CaSO. 4. had been performed in 28 patients. Ceftriaxone was applied as a single local antibiotic in 21 operations and combined with vancomycin in eight operations, and in an additional operation with vancomycin and amphotericin B. Sampling of wound fluid was performed from drains or aspirations. Ceftriaxone concentrations were measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Results. A total of 37 wound fluid concentrations from 16 operations performed in 14 patients were collected. The ceftriaxone concentrations remained approximately within a range of 100 to 200 mg/l up to three weeks. The median concentration was 108.9 mg/l (interquartile range 98.8 to 142.5) within the first ten days. No systemic adverse reactions were observed. Conclusion. Our study highlights new clinical data of locally administered ceftriaxone with CaSO. 4. as carrier material. The near-constant release of ceftriaxone from CaSO. 4. observed in vitro could be confirmed in vivo. The concentrations remained below known local toxicity thresholds. Cite this article: Bone Joint Res 2022;11(11):835–842

Aims. Acetabular edge-loading was a cause of increased wear rates in metal-on-metal hip arthroplasties, ultimately contributing to their failure. Although such wear patterns have been regularly reported in retrieval analyses, this study aimed to determine their in vivo location and investigate their relationship with acetabular component positioning. Methods. 3D CT imaging was combined with a recently validated method of mapping bearing surface wear in retrieved hip implants. The asymmetrical stabilizing fins of Birmingham hip replacements (BHRs) allowed the co-registration of their acetabular wear maps and their computational models, segmented from CT scans. The in vivo location of edge-wear was measured within a standardized coordinate system, defined using the anterior pelvic plane. Results. Edge-wear was found predominantly along the superior acetabular edge in all cases, while its median location was 8° (interquartile range (IQR) -59° to 25°) within the anterosuperior quadrant. The deepest point of these scars had a median location of 16° (IQR -58° to 26°), which was statistically comparable to their centres (p = 0.496). Edge-wear was in closer proximity to the superior apex of the cups with greater angles of acetabular inclination, while a greater degree of anteversion influenced a more anteriorly centred scar. Conclusion. The anterosuperior location of edge-wear was comparable to the degradation patterns observed in acetabular cartilage, supporting previous findings that hip joint forces are directed anteriorly during a greater portion of walking gait. The further application of this novel method could improve the current definition of optimal and safe acetabular component positioning. Cite this article: Bone Joint Res 2021;10(10):639–649

Aim. Fungal periprosthetic joint infections are difficult to treat and often associated with a limited outcome for patients. Candida species account for approximately 90% of all fungal infections. In vivo biofilm models play major role to study biofilm development, morphology, and regulatory molecules for bacteria. However, in vivo modeling of biofilm-associated fungi models are very rare. Furthermore, due to ethical restrictions, mammalian models are replaced with other alternative models in basic research. Recently, we have developed insect infection model G. mellonella larvae to study implant associated biofilm infections with bacteria. This model organism was not used for fungi biofilm infection yet. Thus, we aimed to establish G. mellonella as in vivo model to study fungal implant infections using Candida albicans as model organism and to test anti-fungal medication. Method. Titanium and Stainless steel K-wires were cut into small pieces with size of 4mm. For the infection process, implants were pre-incubated in specified fungal growth culture Candida albicans at 1×10. 7. CFU/ml for 30 min at 150 rpm shaking conditions. Later, these implants were washed with 10ml PBS and implanted in the larvae as mentioned. To analyze the susceptibility of the implant-associated fungal infections towards anti fungal compounds, the larvae were treated with amphotericin B, fluconazole and voriconazole after 24h of implantation. The effect of anti-fungal compounds was measured in terms of survival observation for 5 days and fungal load in larvae on 2. nd. day. To reveal the fungal biofilm formation on implant, the implants were removed on day 3 and processed for SEM analysis. Results. Pre-incubated K-wire caused the Candida infection and observed the death of the larvae. The treatment with antifungal compounds recovered the larvae from the implant-infection, except in case of Voriconazole. However, the recovery with treatment of anti fungal compounds was not effective as the larvae with planktonic infection, which highlights typical biofilm phenotype. Further, the treatment with anti-fungal compounds with Amphotericin B and Fluconazole reduced the fungal load in larvae tissue. The SEM analysis revealed the formation fungal biofilm with hyphae and spores associated with larvae tissue on implant surface. Conclusions. The results from survival analysis, antifungal treatment and SEM analysis are very promising to use of G. mellonella as in vivo model to study fungal infections on implanted materials. Our study highlights the use of G. mellonella larvae as alternative in vivo model to study implant-associated fungal infections that reduces the use of the higher mammals

Introduction and Objective. Bone remodelling is a continuous process whereby osteocytes regulate the activity of osteoblasts and osteoclasts to repair loading-induced microdamage. While many in vitro studies have established the role of paracrine factors (e.g., RANKL/OPG) and cellular pathways involved in bone homeostasis, these techniques are generally limited to two-dimensional cell culture, which neglects the role of the native extracellular matrix in maintaining the phenotype of osteocyte. Recently, ex vivo models have been used to understand cell physiology and mechanobiology in the presence of the native matrix. Such approaches could be applicable to study the mechanisms of bone repair, whilst also enabling exploration of biomechanical cues. However, to date an ex vivo model of bone remodelling in cortical bone has not been developed. In this study, the objective was to develop an ex vivo model where cortical bone was subjected to cyclic strains to study the remodelling of bone. Materials and Methods. Ex vivo model of bone remodelling induced by cyclic loading: At the day of culling, beam-shape bovine bone samples were cut and preserved in PBS + 5% Pen/Strep + 2 mM L-Glut overnight at 37°C. Cyclic strains were applied with a three-point bend system to induce damage with a regime at 16.66 mm/min for 5,000 cycles in sterile PBS in Evolve® bags (maximum strain 6%). A control group was cultured under static conditions. Metabolic activity: Alamar Blue assays were performed after 1 and 7 days of ex vivo culture for each group (Static, Loaded) and normalized to weight. Bone remodelling: ALP activity was assessed in the media at day 1 and 7. After 24 hours cell culture conditioned media (CM) was collected from each group and stored at −80°C. RAW264.7 cells were cultured with CM for 6 days, after which the samples were stained for TRAP, to determine osteoclastogenesis, and imaged. Histomorphometry: Samples were cultured with calcein for 3 days to label bone formation between day 4 and 7. Fluorescent images were captured at day 7. μCT scanning was performed at 3 μm resolution after labelling samples with BaSO. 4. precipitate to quantify bone damage. Results. Bone was sectioned and cultured to maintain live osteoblasts and osteocytes. CM that was obtained 24 hours after cyclic loading and added to RAW264.7 cells cultures, resulted in significantly increased osteoclastogenic potential compared to that from static samples (4.245±1.65% vs 0.88±0.48%, p<0.001). Calcein and HE staining indicated the presence of structures similar to bone remodelling cones in both groups after 7 days of culture. Also, 7 days post-loading, matrix microdamage in the stimulated area, detected with the BaSO. 4. precipitate, were not significantly increased under the load point in loaded samples (0.11±0.05% of bone volume), while at the support areas it was significantly higher (0.2387±0.06%, p<0.001) compared to the static (0.062±0.02%). Conclusions. This study demonstrates that (1) cyclic strains applied on ex vivo bovine cortical bone successfully induced remodelling as characterized by the formation of bone resorption cones, along with an increase of osteoclast formation, and (2) there was an induction of microdamage post loading as shown by the significant increases in microdamage labelled. This supports previous in vivo studies with an increase in osteoclastogenesis up to 7 days post loading. This is the first evidence of the development of an ex vivo model to study osteon remodelling that could be applied to study bone physiology and repair

Articulating cartilage experiences a multitude of biophysical cues. Due to its primary function in distributing load with near frictionless articulation, it is clear that a major stimulus for cartilage homeostasis and regeneration is the mechanical load it experiences on a daily basis. While these effects are considered when performing in vivo studies, in vitro studies are still largely performed under static conditions. Therefore, an increasing complexity of in vitro culture models is required, with the ultimate aim to recreate the articulating joint as accurately as possible. We have for many years utilized a complex multiaxial load bioreactor capable of applying tightly regulated compression and shear loading protocols. Using this bioreactor, we have been able to demonstrate the mechanical induction of human bone marrow stromal cell (BMSC) chondrogenesis in the absence of exogenous growth factors. Building on previous bioreactor studies that demonstrated the mechanical activation of endogenous TGFβ, and subsequent chondrogenesis of human bone marrow derived MSCs, we have been further increasing the complexity of in vitro models. For example, the addition of high molecular weight hyaluronic acid, a component of synovial fluid, culture medium leads to reduced hypertrophy and increased glycosaminoglycan deposition. The ultimate aim of all of these endeavors is to identify promising materials and therapies during in vitro/ ex vivo studies, therefore reducing the numbers or candidates that are finally tested using in vivo studies. This 3R approach can improve the opportunities for success while leading to more ethically acceptable product development pathways

Background. In vivo evaluation of IVD strains is crucial to better understand normal and pathological IVD mechanics, and to evaluate the effectiveness of treatments. This study aimed to 1) develop a novel in vivo technique based on 3T MRI and digital volume correlation (DVC) to measure strains within IVDs and 2) to use this technique to resolve 3D strains within IVDs of healthy volunteers during extension. Methods. This study included 40 lumbar IVDs from eight healthy subjects. The optimal MR sequence to minimise DVC uncertainties was identified by scanning one subject with four different sequences: CISS, T1VIBE, T2SPACE, and T2TSE. To assess the repeatability of the strain measurements in spines with different anatomical and morphological variations four subjects were scanned with the optimal sequence, and uncertainties of the strain measurements were quantified. Additionally, to calculate 3D strains during extension, MRIs were acquired from six subjects in both the neutral position and after full extension. Results. Measurement errors were lowest when using the T2TSE sequence (precision=0.33 ± 0.10%, accuracy=0.48 ± 0.11%). The largest average maximum tensile and shear strains were seen at the L2-L3 level in all volunteers (7.2 ± 1.5% and 6.8 ± 1.1%, respectively), while the L5-S1 level experienced the lowest average tensile and shear strains (3.5 ± 1.0% and 3.9 ± 0.7%, respectively). Conclusion. The findings of this study establish clinical MRI-based DVC (MRI-DVC) as a new tool for in vivo strain measurement within human IVDs. MRI-DVC successfully provided internal strain distributions within IVDs and has great potential to be used for a wide range of clinical applications. Conflict of interest: No conflicts of interest. Source of funding: This work was supported by the EPSRC, New Investigator Award, EP/V029452/1

Aims. Staphylococcus aureus is a major cause of septic arthritis, and in vitro studies suggest α haemolysin (Hla) is responsible for chondrocyte death. We used an in vivo murine joint model to compare inoculation with wild type S. aureus 8325-4 with a Hla-deficient strain DU1090 on chondrocyte viability, tissue histology, and joint biomechanics. The aim was to compare the actions of S. aureus Hla alone with those of the animal’s immune response to infection. Methods. Adult male C57Bl/6 mice (n = 75) were randomized into three groups to receive 1.0 to 1.4 × 10. 7. colony-forming units (CFUs)/ml of 8325-4, DU1090, or saline into the right stifle joint. Chondrocyte death was assessed by confocal microscopy. Histological changes to inoculated joints were graded for inflammatory responses along with gait, weight changes, and limb swelling. Results. Chondrocyte death was greater with 8325-4 (96.2% (SD 5.5%); p < 0.001) than DU1090 (28.9% (SD 16.0%); p = 0.009) and both were higher than controls (3.8% (SD 1.2%)). Histology revealed cartilage/bone damage with 8325-4 or DU1090 compared to controls (p = 0.010). Both infected groups lost weight (p = 0.006 for both) and experienced limb swelling (p = 0.043 and p = 0.018, respectively). Joints inoculated with bacteria showed significant alterations in gait cycle with a decreased stance phase, increased swing phase, and a corresponding decrease in swing speed. Conclusion. Murine joints inoculated with Hla-producing 8325-4 experienced significantly more chondrocyte death than those with DU1090, which lack the toxin. This was despite similar immune responses, indicating that Hla was the major cause of chondrocyte death. Hla-deficient DU1090 also elevated chondrocyte death compared to controls, suggesting a smaller additional deleterious role of the immune system on cartilage. Cite this article: Bone Joint Res 2022;11(9):669–678

Osteoarthritis (OA) is a degenerative joint disease affecting millions worldwide. Early detection of OA and monitoring its progression is essential for effective treatment and for preventing irreversible damage. Although sensors have emerged as a promising tool for monitoring analytes in patients, their application for monitoring the state of pathology is currently restricted to specific fields (such as diabetes). In this study, we present the development of an optical sensor system for real-time monitoring of inflammation based on the measurement of nitric oxide (NO), a molecule highly produced in tissues during inflammation. Single-walled carbon nanotubes (SWCNT) were functionalized with a single-stranded DNA (ssDNA) wrapping designed using an artificial intelligence approach and tested using S-nitroso-N-acetyl penicillamine (SNAP) as a standard released-NO marker. An optical SWIR reader with LED excitation at 650 nm, 730 nm and detecting emission above 1000 nm was developed to read the fluorescence signal from the SWCNTs. Finally, the SWCNT was embedded in GelMa to prove the feasibility of monitoring the release of NO in bovine chondrocyte and osteochondral inflamed cultures (1–10 ng/ml IL1β) monitored over 48 hours. The stability of the inflammation model and NO release was indirectly validated using the Griess and DAF-FM methods. A microfabricated sensor tag was developed to explore the possibility of using ssDNA-SWCNT in an ex vivo anatomic set-up for surgical feasibility, the limit of detection, and the stability under dynamic flexion. The SWCNT sensor was sensitive to NO in both in silico and in vitro conditions during the inflammatory response from chondrocyte and osteochondral plug cultures. The fluorescence signal decreased in the inflamed group compared to control, indicating increased NO concentration. The micro-tag was suitable and stable in joints showing a readable signal at a depth of up to 6 mm under the skin. The ssDNA-SWCNT technology showed the possibility of monitoring inflammation continuously in an in vitro set-up and good stability inside the joint. However, further studies in vivo are needed to prove the possibility of monitoring disease progression and treatment efficacy in vivo. Acknowledgments: The project was co-financed by Innosuisse (grant nr. 56034.1 IP-LS)

Aims. Research on hip biomechanics has analyzed femoroacetabular contact pressures and forces in distinct hip conditions, with different procedures, and used diverse loading and testing conditions. The aim of this scoping review was to identify and summarize the available evidence in the literature for hip contact pressures and force in cadaver and in vivo studies, and how joint loading, labral status, and femoral and acetabular morphology can affect these biomechanical parameters. Methods. We used the PRISMA extension for scoping reviews for this literature search in three databases. After screening, 16 studies were included for the final analysis. Results. The studies assessed different hip conditions like labrum status, the biomechanical effect of the cam, femoral version, acetabular coverage, and the effect of rim trimming. The testing and loading conditions were also quite diverse, and this disparity limits direct comparisons between the different researches. With normal anatomy the mean contact pressures ranged from 1.54 to 4.4 MPa, and the average peak contact pressures ranged from 2 to 9.3 MPa. Labral tear or resection showed an increase in contact pressures that diminished after repair or reconstruction of the labrum. Complete cam resection also decreased the contact pressure, and acetabular rim resection of 6 mm increased the contact pressure at the acetabular base. Conclusion. To date there is no standardized methodology to access hip contact biomechanics in hip arthroscopy, or with the preservation of the periarticular soft-tissues. A tendency towards improved biomechanics (lower contact pressures) was seen with labral repair and reconstruction techniques as well as with cam correction. Cite this article: Bone Joint Res 2023;12(12):712–721

3D printing and Bioprinting technologies are becoming increasingly popular in surgery to provide a solution for the regeneration of healthy tissues. The aim of our project is the regeneration of articular cartilage via bioprinting means, to manage isolated chondral defects. Chrondrogenic hydrogel (chondrogel: GelMa + TGF-b3 and BMP6) was prepared and sterilised in our lab following our standard protocols. Human adipose-derived mesenchymal stem cells were harvested from the infrapatellar fat pad of patients undergoing total knee joint replacements and incorporated in the hydrogel according to our published protocols. The chondrogenic properties of the chondrogel have been tested (histology, immunohistochemistry, PCR, immunofluorescence, gene analysis and 2. nd. harmonic generation microscopy) in vitro and in an ex-vivo model of human articular defect and compared with standard culture systems where the growth factors are added to the media at repeated intervals. The in-vitro analysis showed that the formation of hyaline cartilage pellet was comparable between the two strategies, with a similar metabolic activity of the cells. These results have been confirmed in the ex-vivo model: hyaline-like cartilage was observed within the chondral defect in both the chondrogel group and the control group after 28 days in culture. The use of bioprinting techniques in vivo requires the ability of stem cells to access growth factors directly in the environment they are in, as opposed to in vitro techniques where these factors are provided externally at recurrent intervals. This study showed the successful strategy of incorporating chondrogenic growth factors for the formation of hyaline-like cartilage in vitro and in an ex-vivo model of chondral loss. The incorporation of chondrogenic growth factors in a hydrogel is a possible strategy for articular cartilage regeneration

Cartilage diseases have a significant impact on the patient's quality of life and are a heavy burden for the healthcare system. Better understanding, early detection and proper follow-up could improve quality of life and reduce healthcare related costs. Therefore, the aim of this study was to evaluate if difference between osteoarthritic (OA) and non-osteoarthritic (non-OA) knees can be detected quantitatively on cartilage and subchondral bone levels with advanced but clinical available imaging techniques. Two OA (mean age = 88.3 years) and three non-OA (mean age = 51.0 years) human cadaveric knees were scanned two times. A high-resolution peripheral quantitative computed tomography (HR-pQCT) scan (XtremeCT, Scanco Medical AG, Switzerland) was performed to quantify the bone microstructure. A contrast-enhanced clinical CT scan (GE Revolution Evo, GE Medical Systems AG, Switzerland) was acquired with the contrast agent Visipaque 320 (60 ml) to measure cartilage. Subregions dividing the condyle in four parts were identified semi-automatically and the images were segmented using adaptive thresholding. Microstructural parameters of subchondral bone and cartilage thickness were quantified. The overall cartilage thickness was reduced by 0.27 mm between the OA and non-OA knees and the subchondral bone quality decreased accordingly (reduction of 33.52 % in BV/TV in the layer from 3 to 8 mm below the cartilage) for the femoral medial condyle. The largest differences were observed at the medial part of the femoral medial condyle both for cartilage and for bone parameters, corresponding to clinical observations. Subchondral bone microstructural parameters and cartilage thickness were quantified using in vivo available imaging and apparent differences between the OA and non-OA knees were detected. Those results may improve OA follow-up and diagnosis and could lead to a better understanding of OA. However, further in vivo studies are needed to validate these methods in clinical practice

Aim. To provide proof of concept in an in vivo animal model for the prevention of prosthetic joint infection prevention using electric fields along with conventional antibiotic prophylaxis. Corresponding Author: Marti Bernaus. Method. First, we standardized the animal model to simulate implant contamination during the surgical procedure. We then implanted cobalt-chrome prostheses adapted to both knees of two New Zealand White rabbits, under standard aseptic measures and antibiotic prophylaxis with cefazolin. Prior to implantation, we immersed the prostheses in a 0.3 McFarland inoculum of S. aureus (ATCC 25923) for 30 seconds. In the first animal (control), the joint was directly closed after washing with saline. In the second animal (case), both prostheses were treated with electric current pulses for 30 seconds, washed with saline, and the joint was closed. After 72 hours, both animals were reoperated for the collection of periprosthetic tissue and bone samples, and prosthesis removal. In all samples, we performed quantitative cultures prior to vortexing and sonication, as well as prolonged cultures of the sonication broth. We confirmed the absence of contamination by identification with MALDI-TOF (VITEK-MS) and automated antibiotic susceptibility testing of the isolated colonies (VITEK-2). Results. In the “control” animal, we isolated S. aureus in all studied samples. The bacterial count expressed as log10 (cfu/cm2) in the prostheses of the right and left legs was 9.38 and 8.86, respectively. The bacterial count expressed as log10 (cfu/mL) in bone and periprosthetic tissue biopsies was 2.70 and 2.72 in the right leg and 3.24 and 3.87 in the left leg, respectively. In the “case” animal, where an electric field was applied to the implant after placement in addition to cefazolin prophylaxis, all samples (prosthesis, bone, and periprosthetic tissue) were negative, and no isolation of the inoculated strain of S. aureus was obtained after incubation of the sonication broth for 14 days. Conclusions. This in vivo model suggests the potential effectiveness of applying an electric field to a prosthetic implant in combination with cefazolin for the prevention of PJI development, after exposure of the implant to an inoculum of S. aureus (ATCC 25923). Our findings need to be confirmed using a larger sample size

A novel ex vivo intervertebral disc (IVD) organ model and corresponding sample holder were developed according to the requirements for six degrees of freedom loading and sterile culture in a new generation of multiaxial bioreactors. We tested if the model can be maintained in long-term IVD organ culture and validated the mechanical resistance of the IVD holder in compression, tension, torsion, and bending. An ex vivo bovine caudal IVD organ model was adapted by retaining 5-6 mm of vertebral bone to machine a central cross and a hole for nutrient access through the cartilaginous endplate. A counter cross was made on a customized, circular IVD holder. The new model was compared to a standard model with a minimum of bone for the cell viability and height changes after 3 weeks of cyclic compressive uniaxial loading (0.02-0.2 MPa, 0.2 Hz, 2h/ day; n= 3 for day 0, n= 2 for week 1, 2, and 3 endpoints). Mechanical tests were conducted on the assembly of IVD and holder enhanced with different combinations of side screws, top screws, and bone adhesive (n=3 for each test). The new model retained a high level of cell viability after three weeks of in vitro culture (outer annulus fibrosus 82%, inner annulus fibrosus 69%, nucleus pulposus 75%) and maintained the typical values of IVD height reduction after loading (≤ 10%). The holder-IVD interface reached the following highest average values in the tested configurations: 320.37 N in compression, 431.86 N in tension, 1.64 Nm in torsion, and 0.79 Nm in bending. The new IVD organ model can be maintained in long-term culture and when combined with the corresponding holder resists sufficient loads to study IVD degeneration and therapies in a new generation of multiaxial bioreactors

An ex vivo biomechanical test model for evaluating a novel bone adhesive has been developed. However, at day 1 in the in vivo pilot, high blood flow forced the study to halt until the solution presented here was developed. The profuse bleeding after bone core removal affected the bond strength and was reflected in the lower mean peak value 1.53N. After considering several options, we were successful in sealing the source of blood flow by pressing adhesive into place after bone core removal. After the initial adhesive had cured additional adhesive was used to secure the bone core in place. The animals were sacrificed after 24 h and a tensile test was undertaken on the bone core to failure. The ex vivo study produced mean peak tensile loads of 7.63N SD 2.39N (n=8, 4 rats 8 femurs). Whilst the mean peak tensile loads in the day 1 in vivo pilot were significantly lower 1.53N SD1.57 (n=8, 6 rats 8 femurs − 4 used for other tests). The subsequent layered adhesive bone cores showed a mean peak tensile force of 6.79N SD =3.13 (n=8, 4 rats 8 femurs). 7/8 failed at the bone to glue interface. This is the first successful demonstration of bonding bone in vivo for this class of adhesives. The development of a double adhesive method of fixing a bone core in the distal femur enabled mean peak tensile forces to be achieved in vivo at 24 hours that were comparable with the ex vivo results previously demonstrated. This method supports application in further animal series and over longer time scales. Biomaterials researchers that intend to use gel or paste like preparations in distal femur defects in the rat should be aware of the risks of biomaterial displacement by local blood flow

Articular cartilage has poor repair potential and the tissue formed is mechanically incompetent. Mesenchymal stromal cells (MSCs) show chondrogenic properties and the ability to re-grow cartilage, however a viable human model for testing cartilage regeneration and repair is lacking. Here, we describe an ex vivo pre-clinical femoral head model for studying human cartilage repair using MSCs. Human femoral heads (FHs) were obtained following femoral neck fracture with ethical permission/patient consent and full-depth cartilage wells made using a 3mm biopsy punch. Pancreas-derived mesenchymal stromal cells (P-MSC) were prepared in culture media at ~5000 cells/20µl and added to each well and leakage prevented with fibrin sealant. After 24hrs, the sealant was removed and medium replaced with StemPro. TM. chondrogenesis differentiation medium. The FHs were incubated (37. o. C;5% CO. 2. ) for 3wks, followed by a further 3wks in standard medium with 10% human serum with regular medium changes throughout. Compared to wells with medium only, A-MSCs produced a thin film across the wells which was excised en-block, fixed with 4% paraformaldehyde and frozen for cryo-sectioning. The cell/tissue films varied in thickness ranging over 20-440µm (82±21µm; mean±SEM; N=3 FHs). The thickness of MSC films abutting the cartilage wells was variable but generally greater (15-1880µm) than across the wells, suggesting an attachment to native articular cartilage. Staining of the films using safranin O (for glycosaminoglycans; quantified using ImageJ) was variable (3±8%; mean±SEM; N=3) but in one experiment reached 20% of the adjacent cartilage. A preliminary assessment of the repair tissue gave an O'Driscoll score of 10/24 (24 is best). These preliminary results suggest the ex vivo femoral head model has promise for studying the capacity of MSCs to repair cartilage directly in human tissue, although optimising MSCs to produce hyaline-like tissue is essential. Supported by the CSO (TCS/17/32)

Aims. Extracellular matrix (ECM) and its architecture have a vital role in articular cartilage (AC) structure and function. We hypothesized that a multi-layered chitosan-gelatin (CG) scaffold that resembles ECM, as well as native collagen architecture of AC, will achieve superior chondrogenesis and AC regeneration. We also compared its in vitro and in vivo outcomes with randomly aligned CG scaffold. Methods. Rabbit bone marrow mesenchymal stem cells (MSCs) were differentiated into the chondrogenic lineage on scaffolds. Quality of in vitro regenerated cartilage was assessed by cell viability, growth, matrix synthesis, and differentiation. Bilateral osteochondral defects were created in 15 four-month-old male New Zealand white rabbits and segregated into three treatment groups with five in each. The groups were: 1) untreated and allogeneic chondrocytes; 2) multi-layered scaffold with and without cells; and 3) randomly aligned scaffold with and without cells. After four months of follow-up, the outcome was assessed using histology and immunostaining. Results. In vitro testing showed that the secreted ECM oriented itself along the fibre in multi-layered scaffolds. Both types of CG scaffolds supported cell viability, growth, and matrix synthesis. In vitro chondrogenesis on scaffold showed an around 400-fold increase in collagen type 2 (COL2A1) expression in both CG scaffolds, but the total glycosaminoglycan (GAG)/DNA deposition was 1.39-fold higher in the multi-layered scaffold than the randomly aligned scaffold. In vivo cartilage formation occurred in both multi-layered and randomly aligned scaffolds treated with and without cells, and was shown to be of hyaline phenotype on immunostaining. The defects treated with multi-layered + cells, however, showed significantly thicker cartilage formation than the randomly aligned scaffold. Conclusion. We demonstrated that MSCs loaded CG scaffold with multi-layered zonal architecture promoted superior hyaline AC regeneration. Cite this article: Bone Joint Res 2020;9(9):601–612

Although autografts represent the gold standard for anterior cruciate ligament (ACL) reconstruction, tissue-engineered ACLs provide a prospect to minimize donor site morbidity and limited graft availability. This given study characterizes the ligamentogenesis in embroidered poly(L-lactide-co-ε-caprolactone) (P(LA-CL)) / polylactic acid (PLA) constructs using a dynamic nude mice xenograft model. (P(LA-CL))/PLA scaffolds remained either untreated (co) or were functionalized by gas fluorination (F), collagen foam cross-linked with hexamethylene diisocyanate (HMDI) (coll), or gas fluorination combined with the foam (F+coll). Cell free constructs or those seeded for 1 week with lapine ACL ligamentocytes were implanted into nude mice for 12 weeks. Following explantation, biomechanical properties, cell vitality and content, histopathology of scaffolds (including organs: liver, kidney, spleen), sulphated glycosaminoglycan (sGAG) contents and biomechanical properties were assessed. Implantation of the scaffolds did not negatively affect mice weight development and organs, indicating biocompatibility. All scaffolds maintained their size and shape for the duration of the implantation. A high cell viability was detected in the scaffolds prior to and following implantation. Coll or F+coll scaffolds seeded with cells yielded superior macroscopic properties when compared to the controls. Mild signs of inflammation (foreign-body giant cells, hyperemia) were limited to scaffolds without collagen. Microscopical score values and sGAG content did not differ significantly. Although remaining stable in vivo, elastic modulus, maximum force, tensile strength and strain at Fmax were significantly lower in the in vivo compared to the samples cultured 1 week in vitro, but did not differ between scaffold subtypes, except for a higher maximum force in F+coll compared with F samples (in vivo). Scaffold functionalization with fluorinated collagen foam provides a promising approach for ACL tissue engineering. (shared first authorship). Acknowledgement: The study was supported by DFG grants SCHU1979/9-1 and SCHU1979/14-1